Scaffolded nucleic acid polymer particles and methods of making and using

ABSTRACT

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

This is a continuation-in-part of U.S. patent applications Ser. Nos.12/474,897 and 12/475,311 both filed 29 May 2009, and claims priorityunder U.S. provisional applications Ser. No. 61/263,734 filed 23 Nov.2009; Ser. No. 61/291,788 filed 31 Dec. 2009; and Ser. No. 61/297,203filed 21 Jan. 2010. All of the foregoing applications are incorporatedby reference in their entireties.

BACKGROUND

In order to generate sufficient signal for analysis, many applicationsin genomics and biomedical research require the conversion of nucleicacid molecules in a library into separate, or separable, libraries ofamplicons of the molecules, e.g. Margulies et al, Nature 437: 376-380(2005); Mitra et al, Nucleic Acids Research, 27: e34 (1999); Shendure etal, Science, 309: 1728-1732 (2005); Brenner et al, Proc. Natl. Acad.Sci., 97: 1665-1670 (2000); and the like. Several techniques have beenused for making such conversions, including hybrid selection (e.g.,Brenner et al, cited above); in-gel polymerase chain reaction (PCR)(e.g. Mitra et al, cited above); bridge amplification (e.g. Shapero etal, Genome Research, 11: 1926-1934 (2001)); and emulsion PCR (cmPCR)(e.g. Margulies et al, cited above). Most of these techniques employparticulate supports, such as beads, which spatially concentrate theamplicons for enhanced signal-to-noise ratios, as well as otherbenefits, such as, better reagent access.

These techniques have several drawbacks. In some cases, amplicons areeither in a planar format (e.g. Mitra et al, cited above; Adessi et al,Nucleic Acids Research, 28: e87 (2000)), which limits ease ofmanipulation and/or reagent access, or the amplicons are on beadsurfaces, which lack sufficient fragment density or concentration foradequate signal-to-noise ratios. In other cases, amplifications must bedone in emulsions in order to obtain clonal populations of templates.Such emulsion reactions are labor intensive and require a high degree ofexpertise, which significantly increases costs. It would be very usefulif supports were available which were capable of providing a higherdensity of analyte binding or attachment sites, particularly for clonalpopulations of nucleic acid fragments. It would also be advantageous ifsuch supports did not require emulsion reactions for producing clonalpopulations.

Gels have been widely used as supports in analytical and syntheticprocesses and as encapsulating agents, e.g. Weaver et al, U.S. Pat. No.5,055,390; Tmovsky et al, U.S. Pat. No. 6,586,176, and have interiorsaccessible to analytical reagents. However, such particulates arelimited in that they are typically produced with widely varying sizedistributions, particularly at lower size ranges, e.g. less than about30 μm, which makes them unsuitable for many exacting analyticalapplications, such as large scale DNA sequencing.

It would be highly useful if methods and compositions were available forcreating small-sized monodisperse populations of gel-based particulatesupports, which could be readily loaded with analytes, such as ampliconsof nucleic acid fragments.

SUMMARY OF THE INVENTION

The present invention is generally directed to particle compositions fornucleic acid analysis, which address the aforementioned issues withcurrent methodologies, as well as other related issues. The presentinvention is exemplified in a number of implementations andapplications, some of which are summarized below and throughout thespecification.

In one aspect, the invention includes the production and use of porousmicroparticles for increasing the number of polynucleotides templateswithin a defined volume. In one embodiment such porous microparticlescomprise three-dimensional scaffolds for attaching greater numbers oftemplate molecules than possible with solid beads that have only atwo-dimensional surface available for attachment. In one embodiment,such porous microparticles are referred to herein as nucleic acidpolymer particles.

In another embodiment, such porous microparticles comprise particleshaving shapes with larger surface to volume ratios than sphericalparticles. Such shapes include tubes, shells, hollow spheres withaccessible interiors (e.g. nanocapsules), barrels, multiply connectedsolids, including doubly connected solids, such as donut-shaped solidsand their topological equivalents, triply connected solids and theirtopological equivalents, four-way connected solids and theirtopologically equivalents, and the like. Such porous microparticles arereferred to herein as “non-spheroidal microparticles.” Techniques forproducing and characterizing such particles are disclosed in Elaissari,editor, Colloidal Polymers: Synthesis and Characterization (MarcelDekker, Inc., New York, 2003), and like references.

In another aspect the invention provides a composition of nucleic acidpolymer particles each comprising polynucleotides attached to anon-nucleosidic polymer network, each such polymer network having avolume and the polynucleotides being attached to the polymer networkthroughout its volume, wherein the number of attached polynucleotides isat least 6.9×10⁴ per μm³ and wherein the oligonucleotides have anaverage nearest neighbor distance of 22 nm or less. In one aspect, thepolynucleotide is a DNA fragment in the range of from 100 to 500nucleotides in length, or in the range of from 100 to 200 nucleotides inlength. In another aspect, such polynucleotide is a double stranded DNA(dsDNA) having a length in the range of from 150 to 250 basepairs.

In another aspect, the invention provides amplicon libraries, suchlibraries comprising a plurality of amplicons, each amplicon comprisinga clonal population of a single polynucleotide from a nucleic acidlibrary, each polynucleotide of the clonal population being attached toa non-nucleosidic polymer network, each such polymer network having avolume and the polynucleotides of the clonal population being attachedto the polymer network throughout its volume, wherein the number ofattached polynucleotides is at least 6.9×10⁴ per μm³. In another aspect,polynucleotides of such amplicons have an average nearest neighbordistance of 22 nm or less, or an average nearest neighbor distance of 20nm or less. In still another aspect, such polynucleotides are each adouble stranded DNA (dsDNA) having a length in the range of from 150 to250 basepairs, or a length in the range of from 150 to 200 basepairs.

In one aspect, an amplicon library of the invention comprises aplurality of amplicons, each amplicon comprising a clonal population ofa single polynucleotide from a nucleic acid library, each polynucleotideof the clonal population being attached to a non-nucleosidic polymernetwork, each such polymer network having a volume and thepolynucleotides of the clonal population being attached to the polymernetwork throughout its volume, wherein the number of attachedpolynucleotides is at least 6.9×10⁴ per μm³. In one embodiment, aplurality of amplicons is in the range of from 10⁴ to 10⁷ amplicons.

In another aspect, the invention provides methods of making monodispersepopulations of gel particles by combining a monodisperse emulsion of agel reaction mixture without an initiator and an emulsion with adispersed phase containing an initiator or a continuous phase solutionsaturated with an initiator. In one embodiment, volumes of the gelparticles of such monodisperse populations have coefficients ofvariation of less than fifteen percent, or in another embodiment, lessthan twelve percent.

In another aspect, the invention provides a method of making ampliconlibraries comprising the steps: (a) combining in an amplificationreaction mixture a library of polynucleotide fragments each having atleast one primer binding site and a population of non-nucleosidicpolymer networks, each such polymer network having a volume of less than1.4×10⁴ μm³ and having primers attached thereto, and the volumes of thenon-nucleosidic polymer networks having a coefficient of variation offifteen percent or less; (b) performing an amplification reaction sothat primers of the polymer networks are each extended along apolynucleotide fragment annealed thereto so that clonal populations ofsuch polynucleotide fragments are formed on the polymer networks,thereby forming an amplicon library. In one embodiment, the method ofmaking amplicon libraries further includes a step of enriching polymernetworks having clonal populations of polynucleotide fragments attachedby separating them from polymer networks without such fragments. Inanother embodiment, such separation is accomplished by affinityseparation or by electrophoretic separation.

In still another aspect, the invention includes methods of usingmonodisperse gel particle compositions to make amplicon librarieswithout an emulsion reaction.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the presence of nucleic acid polymer particles insidemicrowells of a semiconductor sequencing device and the effects ofdifferent polymer network sizes within a library.

FIG. 2A schematically illustrates production of spheroidal gel particlesby membrane emulsification using a micromachined membrane and continuouspolymerization by heat.

FIG. 2B schematically illustrates another embodiment for producingspheroidal gel particles by membrane emulsification and batch modepolymerization by heat.

FIG. 3 diagrammatically illustrates a bridge PCR on a surface.

FIG. 4 diagrammatically illustrates bridge PCR on a suspension ofnucleic acid polymer particles.

FIG. 5 illustrates a method of minimizing cross-contamination of bridgePCR templates among closely packed particles.

FIG. 6 illustrates a method of using adaptor oligonucleotides to allowone kind of nucleic acid polymer particle to be used to capture andamplify a selected set of nucleic acids, such as particular exons of oneor more genes.

FIG. 7 shows a simple thermocycler for carrying out PCRs whilepreventing porous particles from settling at the bottom of a reactionvessel.

FIG. 8 illustrates steps of a nucleic acid sequencing method usingnucleic acid polymer particles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, molecular biology (including recombinant techniques), cellbiology, and biochemistry, which are within the skill of the art. Suchconventional techniques include, but are not limited to, preparation ofsynthetic polynucleotides, polymerization techniques, chemical andphysical analysis of polymer particles, nucleic acid sequencing andanalysis, and the like. Specific illustrations of suitable techniquescan be had by reference to the example herein below. However, otherequivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Hermanson, Bioconjugate Techniques, Second Edition (Academic Press,2008); Merkus, Particle Size Measurements (Springer, 2009); Rubinsteinand Colby, Polymer Physics (Oxford University Press, 2003); and thelike.

The invention is directed to methods and compositions for enhancing thesensitivity of nucleic acid analysis, particularly where many differentnucleic acid fragments are assayed simultaneously, such as inlarge-scale parallel DNA sequencing reactions. In one aspect, thecompositions of the invention result from the conversion of a library ofindividual nucleic acid fragments into a library of individual solidphase amplicons that provide higher concentrations of fragments peramplicon and greater uniformity of amplicon size than currentmethodologies. Solid phase amplicons of the invention are compositematerials made up of a framework, or scaffold, of a hydrophilic covalentnon-nucleosidic polymer (referred to herein as a “polymer network” or asa “porous microparticle”) and covalently attached copies of usually onekind of nucleic acid fragment, or in some embodiments, two kinds ofnucleic acid fragment. In some preferred embodiments, such nucleic acidfragment is a nucleic acid primer. In other preferred embodiments, suchnucleic acid fragment is a DNA fragment from a library, which may havebeen formed on a polymer network by extension of a covalently attachedprimer. (In such embodiments, it is understood that a single “kind” inthis case, may include unextended primers or partially extended primers,which have different lengths but otherwise identical sequences). Suchsolid phase amplicons are synonymously referred to as “scaffoldednucleic acid polymer particles” or simply “nucleic acid polymerparticles.” Compositions of the invention include libraries orcollections of such solid phase amplicons, or equivalently, nucleic acidpolymer particles. In one aspect, the invention includes compositionscomprising populations of such solid phase amplicons. In one aspect,polymer networks are stable in a wide pH range, e.g. from 4 to 10, andespecially from 6 to 9, and they are chemically and physically stable inphysiological salt solutions and/or electrolytes. Likewise, polymernetworks are preferably inert to reactants, reagents and/or reactionconditions and buffers used in analytical assays and reactions fornucleic acids, including, but not limited to, polymerase reactions,ligase reactions, nuclease reactions, polymerase chain reactions, andthe like. Polymer networks are preferably chemically and physicallystable over a wide temperature range, e.g. 0° C. to 100° C., and 5° C.to 95° C. In one aspect, polymer networks are substantially nonswellingin under a wide range of reaction conditions, particularly polymeraseextension reaction conditions. In one aspect, by substantiallynonswelling, it is meant that the volume of a polymer network changes byno more than five percent within a temperature range of from 25° C. to70° C. and under chemical conditions of physiological or assay salt andpH in the range of from 6 to 10, and especially in the range of from 7to 9. In one aspect, the porosity of polymer networks permits free orsubstantially free diffusion of proteins having a size in the range offrom 50 to 200 kilodaltons, or from 50 to 150 kilodaltons, or from 50 to125 kilodaltons. In another aspect, the porosity of polymer networkspermits free or substantially free diffusion of nucleic acidpolymerases. In various embodiments, such porosity may be selected topermit free or substantially free diffusion of primers and/or a DNApolymerase, including but not limited to, a Taq polymerase, a 9° Npolymerase, an E. coli DNA polymerase I, a T7 DNA polymerase, a Bsu DNApolymerase, a Klenow fragment DNA polymerase, a Phusion DNA polymerase,a Vent DNA polymerase, a Bst DNA polymerase, a phi29 DNA polymerase, aT4 DNA polymerase, or the like. In another embodiment, polymer networkshave a porosity that renders them permeable to proteins having a size inthe range of from 50 to 200 kilodaltons, or from 50 to 150 kilodaltons,or from 50 to 125 kilodaltons. In one embodiment, the porosity ofpolymer networks is selected so that such permeability is at least fiftypercent of the diffusability in polymer-free solution, or at leasttwenty-five percent of such diffusability, or at least ten percent ofsuch diffusability. In another aspect, polymer networks have an averagepore size in the range of from 20 to 200 nm in diameter, or from 25 to100 nm in diameter, or from 30 to 100 nm in diameter.

In another aspect, polymer networks may have empty channels or hollowcores that when taken together with the polymer network volume compriseat least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% of the combinedvolume.

In another aspect, nucleic acid fragments are uniformly attachedthroughout the volume of polymer networks. In some embodiments, nucleicacid fragments are uniformly and randomly attached throughout the volumeof polymer networks (i.e. as approximately a Poisson distribution). Inyet other embodiments, nucleic acid fragments may be attached throughouta layer or portion of a polymer network. In further embodiments, nucleicacid fragments may be attached non-uniformly throughout the volume ofpolymer networks. For example, in spherically shaped polymer networks, aconcentration of attached nucleic acid fragments may be a function ofdistance from the center of such polymer network. In one suchembodiment, such function describes a monotonically decreasingconcentration from the surface of a polymer network to its center.

The density of nucleic acids may be expressed in terms of expected oraverage nearest neighbor distance, which allows surface densities to becompared with volume densities. Equivalent densities of nucleic acidsdistributed throughout a spheroidal volume have larger expected nearestneighbor distances than those of nucleic acids distributed on thesurface of such a volume. Expected nearest neighbor distances forPoisson distributed points or molecules are readily computed forsurfaces or volumes, e.g. Pielou, Introduction to Mathematical Ecology(Wiley-Interscience, New York, 1977). Since large molecules or molecularcomplexes of interest (e.g. template-primer-polymerase complexes) havevolumes roughly in the range of from 1000 to 1.2×10⁵ nm³ (Holmes et al,Electrophoresis, 12: 253-263 (1991)), higher concentrations of such canbe achieved by attaching them throughout a volume rather than on asurface. In one aspect, nucleic acid polymer particles of the inventionhave 150 to 200 basepair nucleic acids immobilized throughout aspheroidal volume having a diameter in the range of from 0.5 to 10 μm inan approximate Poisson distribution having an expected nearest neighbordistance in the range of from 15 to 22 nm. In another aspect, suchspheroidal volume has a diameter in the range of from 1 to 10 μm andsuch Poisson distribution of dsDNAs has an expected nearest neighbordistance in the range of from 18 to 20 nm.

Polymer networks may have a variety of shapes, including but not limitedto, spherical, cylindrical, barrel shaped, toroidal, conical, tubular,hemispherical, cubical, and topological equivalents of the foregoing. Inone aspect, polymer networks are spherically shaped, which are readilyobtained from emulsion-based methods of making them. An importantapplication of nucleic acid polymer particle compositions is their usein massively parallel sequencing reactions where nucleic acids attachedto the polymer networks making up such particles are derived fromfragments of a target polynucleotide of interest, such as a genome. Inseveral large scale sequencing approaches, clonal populations offragments, usually attached to separate beads, are subjected tosequencing reactions in microwells, or equivalent enclosures, e.g. asdisclosed in Rothberg et al, U.S. patent publication 2009/0127589; Gamalet al, U.S. Pat. No. 7,595,883; Leamon et al, U.S. Pat. No. 7,323,305;and the like. In particular, approaches based on making electrochemicalmeasurements, such as Rothberg et al (cited above), benefit frompopulations of nucleic acid polymer particles that have low coefficientsof variation. For example, FIGS. 1A and 1B illustrate top views of ISFETsensor arrays (100), e.g. as disclosed in Rothberg et al (cited above),which include a rectilinear array of microwells (102) filled withmicroparticles (104) and electrolyte (106). In FIG. 1A, the coefficientof variation of the diameters of microparticles (104) is 51%, and inFIG. 1B the corresponding coefficient of variation is 22%. Wheneverelectrical measurements are made through or across the microwells,differences in microparticle sizes and motions give rise to resistivenoise; thus, the greater the coefficient of variation in microparticlesize, the greater the noise. Generally, if the coefficient of variationof the size of a particle is large, several difficulties can arise: (i)flows of reagents across the beads or particles may dislodge and washaway some particles, e.g. small particles or particles too large tocompletely fit into a microwell, (ii) in the case of ion-basedsequencing approaches, such as pH-based sequencing disclosed in Rothberget al (cited above), fluid noise sensed by a electronic-based sensorwill vary depending on the fluid gap around the particle in the well,and (iii) signals generated by reactions taking place on the nucleicacids of a particle will vary with the size of the particle, which addsa signal processing complication because many sequencing chemistriesgenerate a signal that depends on the number of bases incorporated in apolymerase extension reaction. As a consequence, it is advantageous tohave populations of nucleic acid polymer particles with sizes, e.g.volumes, or diameters for spherical particles, which have a lowcoefficient of variation. In one aspect, such populations of particleshave volumes with coefficients of variation less than 25 percent; and infurther aspects, such populations of particles have volumes withcoefficients of variation less than 20 percent; and in further aspects,such populations of particles have volumes with coefficients ofvariation less than 15 percent; and in further aspects, such populationsof particles have volumes with coefficients of variation less than 10percent; or less than 5 percent. Preferably, populations of sphericalpolymer networks have coefficients of variation less than 15 percent,and more preferably, less than 10 percent, or less than 5 percent.

In another aspect, spherical polymer networks have average sizes(diameters) less than 30 μm, or in the range of from 0.5 μM to 30 μm, orin the range of from 0.5 μm to 15 μm, or in the range of from 0.5 μm to10 μm.

In some embodiments, the porous microparticle is hollow (i.e., it has ahollow core); while in other embodiments it has a porous core.

As mentioned above, an aspect of the invention includes ampliconlibraries comprising a plurality of solid phase amplicons each, in turn,comprising nucleic acid polymer particles each having a clonalpopulation of polynucleotides, such as genomic fragments, attachedthroughout the volume of a polymer network.

Compositions

Polymer networks may be made of a wide variety of components and themethod of manufacturing may vary widely. Design factors for makingpolymer networks include, but are not limited to, the following: (i) thepolymers of the networks are hydrophilic, (ii) they are capable ofhaving a pore and/or network structure (e.g. average pore diameter,tortuosity, and the like) that permits interior access to variousenzymes, especially polymerases, (iii) they are physically andchemically stable under conditions where biomolecules, such as enzymes,are functional and they are substantially non-swelling under the sameconditions. There is a great amount of guidance in the art for selectingpolymers and polymerization methodologies to produce polymer networksmeeting such performance criteria, such as the following exemplaryreferences, which are incorporated by reference: Saltzman and Langer, J.Biophys., 55:163 (1989); Ghosh et al, U.S. Pat. No. 5,478,893;Mirzabekov, U.S. Pat. No. 6,656,725; Johnson et al, U.S. Pat. No.6,372,813; Tang and Xiao, Biosensors and Bioelectronics, 24: 1817-1824(2009); Boles et al, U.S. Pat. Nos. 5,932,711 and 6,180,770; Xiao et al,Electrophoresis, 28: 1903-1912 (2007); Holmes et al, Electrophoresis,12: 253-263 (1991); Shapero et al, Genome Research, 11: 1926-1934(2001); Righetti et al, J. Biochem. Biophys. Methods, 4: 347-363 (1981);Mitra et al, Nucleic Acids Research, 27: e34 (1999); Rehman et al,Nucleic Acids Research, 27: 649-655 (1999); Smith, U.S. Pat. No.4,485,224; Chiari et al, U.S. Pat. No. 5,785,832; Rickwood and Hames,Editors, Gel Electrophoresis of Nucleic Acids (IRL Press, Oxford, 1982);Chrambach, The Practice of Quantitative Gel Electrophoresis (VCH,Deerfield Beach, 1985); Mitra et al, Anal. Biochem., 320: 55-65 (2003);Kenney et al, Biotechniques, 25: 516 (1998); Elaissari, editor,Colloidal Polymers: Synthesis and Characterization (Marcel Dekker, Inc.,New York, 2003); and the like.

In one aspect, polymer networks comprise polymers selected from thefollowing group: agarose; polyoxybutylene; dimethylacrylamide;polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; and the like. Asdescribed more fully below, such polymers may be formed into polymernetworks using conventional methodologies, e.g. cross-linking methods,methods for producing desired shapes, and the like.

In some embodiments, the nucleic acids are bound to polymer networkswith one or more non-nucleic acid polymers or linking groups. In someembodiments, the non-nucleic acid polymers are polyethylene glycol (PEG)polymers. The PEG polymers may be of varying lengths. In someembodiments, the non-nucleic acid polymers are dextran polymers and/orchitosan polymers. In some embodiments, the non-nucleic acid polymersinclude PEG polymers and dextran polymers. In some embodiments, thenon-nucleic acid polymers include PEG polymers and chitosan polymers.The non-nucleic acid polymers may be linear or branched. Still othermethods for attaching nucleic acids to beads are taught by Lund et al.,Nucleic Acids Research, 1988, 16(22):10861-10880, Joos et al. AnalBiochem, 1997, 247:96-101, Steinberg et al. Biopolymers, 2004,73:597-605, and Steinberg-Tatman et al. Bioconjugate Chem 200617:841-848.

In one embodiment, nucleic acid polymer particles are made by firstmaking polymer networks that incorporate either bromoacetyl groups oralternative thiol groups, then reacting either a thiol derivatizedoligonucleotide or a bromoacetyl-derivatized oligonucleotiderespectively, as taught by Ghosh et al, U.S. Pat. No. 5,478,893, whichis incorporated by reference. Synthesizing bromoacetyl-derivatized andthiol-derivatized oligonucleotides is further disclosed by Gryaznov,U.S. Pat. No. 5,830,658, which is incorporated by reference. In oneaspect, polyacrylamide particles are employed that may be size selectedeither before or after bromoacetyl- and thiol-derivatized components arereacted.

In another embodiment, nucleic acid polymer particles are made bypreparing a polymer network that incorporates a click chemistryfunctionality then combining it with oligonucleotides having acomplementary click chemistry functionality, so that rapid and specificbonds are formed and a nucleic acid polymer particle results. Clickchemistry functionalities and reactions are well-known and are disclosedin the following references, which are incorporated by reference:Lahann, editor, Click Chemistry for Biotechnology and Material Science(Wiley, 2009); Kolb et al, Angew. Chem. Int. Ed., 40: 2004-2021 (2001);Binder et al, Macromolecular Rapid Comm., 28: 15-54 (2007); Sharpless etal, U.S. Pat. No. 7,375,234; Carell et al, U.S. patent publication2009/0215635; and the like. Reagents containing click chemistry reactivefunctionalities and complementary functionalities are commerciallyavailable from Glen Research (Sterling, Va.); Sigma Aldrich (St. Louis,Mo.), baseclick GmbH (Tutzing, Germany); and like companies. In oneaspect, the click chemistry reactive functionality is an azide and theclick chemistry complementary functionality is an alkene. In oneembodiment, a reaction between such functionalities is catalyzed bycopper(I). In another aspect, a click chemistry reactive functionalityor complementary functionality is incorporated into a polyacrylamidepolymer matrix.

Of particular interest are polymer networks comprising polyacrylamidegels. Polyacrylamide gels are formed by copolymerization of acrylamideand bis-acrylamide (“bis,” N,N′-methylene-bisacrylamide). The reactionis a vinyl addition polymerization initiated by a freeradical-generating system. Polymerization is initiated by ammoniumpersulfate and TEMED (tetramethylethylenediamine): TEMED accelerates therate of formation of free radicals from persulfate and these in turncatalyze polymerization. The persulfate free radicals convert acrylamidemonomers to free radicals which react with unactivated monomers to beginthe polymerization chain reaction. The elongating polymer chains arerandomly crosslinked by bis, resulting in a gel with a characteristicporosity which depends on the polymerization conditions and monomerconcentrations. Riboflavin (or riboflavin-5′-phosphate) may also be usedas a source of free radicals, often in combination with TEMED andammonium persulfate. In the presence of light and oxygen, riboflavin isconverted to its leuco form, which is active in initiatingpolymerization, which is usually referred to as photochemicalpolymerization. In a standard nomenclature for forming polyacrylamidegels, T represents the total percentage concentration (w/v, in mg/mL) ofmonomer (acrylamide plus crosslinker) in the gel. The term C refers tothe percentage of the total monomer represented by the crosslinker. Forexample, an 8%, 19:1 (acrylamide/bisacrylamide) gel would have a T valueof 8% and a C value of 5%.

In one aspect, polymer networks comprise polyacrylamide gels with totalmonomer percentages in the range of from 3-20 percent, and morepreferably, in the range of from 5 to 10 percent. In one embodiment,crosslinker percentage of monomers is in the range of from 5 to 10percent. In a particular embodiment, polymer networks comprise 10percent total acrylamide of which 10 percent is bisacrylamide.

Accordingly, in one aspect, the invention includes a polyacrylamideparticle composition comprising a population of polyacrylamide particleswith an average particle size of less than 15 μm with a coefficient ofvariation of less than 15 percent. In one embodiment, the polyacrylamideparticles have a weight:volume percentage of twenty-five percent orless. In another embodiment, the polyacrylamide particles are spheroidaland have an average diameter of less than 15 μm with a coefficient ofvariation of less than 15 percent.

Methods of Making Nucleic Acid Polymer Particles

Nucleic acid polymer particles of the invention may be made by a widevariety of methods. In one aspect, such method include steps of (i)forming a reaction mixture whose polymerization may be controlled byphysical conditions, e.g. heat, or the addition of a catalyst; (ii)performing a polymerization reaction to produce polymer networks orcandidate polymer networks or nucleic acid polymer particles orcandidate polymer particles depending on reactants and conditionsemployed, and (iii) optionally, selecting candidate polymer networks orcandidate nucleic acid polymer particles in a predetermined size range.Nucleic acid polymer particles may be made by first making polymernetworks followed by attachment of polynucleotides, or they may be madeby co-polymerization of oligonucleotide components along with monomersand crosslinkers. In addition to the chemical processes that determinethe composition of polymer networks and nucleic acid polymer particles,physical process are employed to create such networks and particles withdesired shapes and size distributions. Such physical processes include,but are not limited to, flow focusing using microfluidics devices, e.g.Nisisako et al, LabChip, 8: 287-293 (2008); Kumaresan et al, Anal.Chem., 80: 3522-3529 (2008), pneumatic disruption of a sheath-sampleflow stream, e.g. Lin et al, Biomed Microdevices, 9: 833-843 (2007);sieving, molding, e.g. Rolland et al, J. Am. Chem. Soc., 127:10096-10100 (2005), sonication, controlled shearing, and membraneemulsion. Further exemplary references include the following: Mak et al.Adv. Funct. Mater. 2008 18:2930-2937; Morimoto et al. MEMS 2008 TucsonAriz. USA Jan. 13-17, 2008 Poster Abstract 304-307; Lee et al. Adv.Mater. 2008 20:3498-3503; Martin-Banderas et al. Small. 20051(7):688-92; and published PCT application WO03/078659. Of particularinterest are the following three methods of forming polymer networks.

UV polymerization. Polymer networks may be made by polymerization ofacrylamide spray droplets generated by single or multiple nozzleslocated on an oscillating membrane, such as in a commercially availablesystem from The Technology Partnership (www.ttp.com) which spraysdroplets from single or multiple nozzles located on a stainless steelmembrane by piezo electronically actuating the membrane and allowing itto oscillate at its natural resonance frequency. This yieldsmonodispersed droplets in a gaseous atmosphere (such as Argon) at ratesof tens of thousands to millions of droplets per second. These dropletsare then streamed passed a strong UV light source for photoinitiatedpolymerization.

Polymerization with molding. This approach involves the molding of apaste which disperses the acrylamide, bisacrylamide and acrydite labeledoligonucleotides in a sacrificial “porogen” followed by, but not limitedto, photoinitiated radical polymerization of the acrylamide monomerswith subsequent removal of the porogen my dissolution and repeatedwashing. The molding technology is available through LiquidiaTechnologies (Research Triangle Park, N.C.) and disclosed in DeSimone etal, PCT publication WO 2007/024323, and like references. Such approachedare particularly useful for producing non-spheroidal microparticles indefined shapes, such as tetrahedral shapes, hemispherical shapes, barrelshapes, open capsular shapes, toroidal shapes, tube shapes, and thelike, which have greater surface areas than spheroidal shaped particleswith the same solid volume. Preferably, the areas of the non-spheroidalmicroparticles in a composition are substantially the same. In oneaspect, substantially the same in reference to non-spheroidalmicroparticles means that the areas of the microparticles in acomposition have a coefficient of variation of less than 15 percent, orless than 10 percent, or less than 5 percent. In one embodiment,non-spherical microparticles of the invention have at least twice thesurface area of a sphere with equal volume, or preferably, at leastthree times the surface area, or at least four time the surface area, orat least five times the surface area: In another embodiment,non-spheroidal microparticles are composed of polyacrylamide gel. Instill another embodiment such polyacrylamide gel is made using acrylditeoligonucleotides so that the resulting non-spheroidal microparicles havecovalently attached oligonucleotides, which may be used as primers inextension reactions, ligation reactions, amplification reactions, or thelike. Alternatively, oligonucleotides or other reagents, such asantibodies, may be attached by using linking groups and chemistriesknown in the art, such as described above. In further preference,non-spheroidal microparticles are compact in that they may be closelyenclosed within a microwell or other reaction chamber. In oneembodiment, non-spheroidal microparticles of the invention may beenclosed by a sphere having a volume twice that of the non-spheroidalmicroparticle, or a volume three times that of such microparticle, orfour time that of such microparticle. In another embodiment,non-spheroidal microparticles of the invention are enclosed by acylinder having a diameter:height aspect ratio of 1:1.5 and a diameterof 10 μm, or 5 μm, or 2 μm, or a cylinder having a diameter:heightaspect ratio of 1:1 and a diameter of 10 μm, or 5 μm, or 2 μm, or 1 μm.

Membrane emulsification. Polymerization of emulsified acrylamiderequires a) control of particle size distribution during polymerization,and b) a controllable polymerization method. Control of sizedistribution requires both the minimization of polydispersity due to theemulsification process as well as minimization of instability of theemulsion leading to coalescence of individual drops prior topolymerization. Highly monodisperse emulsions may be achieved throughmicrosieve emulsification techniques (such as provided commercially byNanomi B.V., The Netherlands) and disclosed the following exemplaryreferences: Wissink et al, PCT publication WO2005/115599; Nakajima etal, U.S. Pat. No. 6,155,710; Qiu et al, U.S. Pat. No. 7,307,104;Gijsbertsen-Abrahase, “Membrane emulsification: process principles,”(Ph.D. Thesis, Wageningen Universiteit, 2003); Geerken, “Emulsificationwith micro-engineered devices”, Ph.D. Thesis, University of Twente,ISBN: 90-365-2432-6, 1974; Yuan, et.al., “Manufacture of controlledemulsions and particulates using membrane emulsification”, Desalination,224, 2008; Geerken, et.al., “Interfacial aspects of water drop formationat micro-engineered orifices”, Journal of Colloid and Interface Science,312, 2007; Sotoyama, et.al., “Water/Oil emulsions prepared by themembrane emulsification method and their stability”, Journal of FoodScience, Vol. 64, No 2, 1999; Kosvintsev, et.al., “Membraneemulsification: Droplet size and uniformity in the absence of surfaceshear”, Journal of Membrane Science, 313, 2008; Egidi, et.al., “Membraneemulsification using membranes of regular pore spacing: Droplet size anduniformity in the presence of surface shear”, Journal of MembraneScience, 323, 2008; Abrahamse, et.al., “Analysis of droplet formationand interactions during cross-flow membrane emulsification”, Journal ofMembrane Science, 204, 2002; Katoh. et.al., “Preparation of foodemulsions using a membrane emulsification system”, Journal of MembraneScience, 113, 1996; Charcosset, et.al., “The membrane emulsificationprocess—a review”, Journal of Chemical Technology and Biotechnology, 79,209-218, 2004; and the like.

In membrane-based emulsification to produce particles, a discontinuousphase (aqueous solution of monomers and other components) is extrudedthrough a plate with multiple through holes where the other side of theplate is constantly washed with a stream of continuous phase (oilformulation with surfactant) such that the droplets that break off fromthe individual orifices are carried away by a stream of continuousphase. The droplet stream is then passed through a heated section oftubing which will initiate the polymerization and is finally collectedin bulk for extraction of the polymer particles.

FIG. 2A illustrates apparatus in one approach to membraneemulsification. Aqueous phase gel reaction mixture (200) is passedthrough membrane (208) held in reaction vessel (210). Membrane (208) haspores or orifices (209) that create droplets (211) of a predeterminedsize (as shown in expanded view (212)) dispersed in an immisciblecontinuous phase fluid (202). As droplets (211) are formed, slowlyflowing continuous phase (222) sweeps them away from membrane (208) totubing (214) where (in this embodiment) droplets (211) are polymerizedby applying heat (216). The flow of continuous phase (202) may becontrolled by syringe pump (204). The length and diameter of tubing(214) is selected to correspond to the amount of time required forpolymerization given the amount of heat applied. Polymerized droplets(218) are deposited in collection vessel (220), after which they areremoved and washed to remove traces of continuous phase (202). Thevolume of the aqueous phase gel reaction mixture (200) is a relativelysmall (200 ul-5 ml) and is pumped at low flow rates (200 uL/hr to 1mL/hr) such that the rate of droplet formation at each orifice (209) isabout 2-20 drops/second. A syringe pump or gravity flow may be used forthis purpose. Continuous phase (202), an oil/surfactant formulation is amuch larger volume, e.g. 100-1000× greater than the aqueous flow. Alarge syringe pump (204) or pressure driven flow (pneumatic) may be usedto control its flow and volume. In this embodiment, as mentioned above,the stream containing an emulsion of aqueous drops in oil/surfactantformulation passing through tubing (214) is passed through a heatedsection of tubing (the I.D. of the tubing may be increased to reduce thelinear flow rate, backpressure and increase the residence time in theheated section. The polymerized particles are finally collected incollection vessel (220) for extraction of the polymer particles.

In one aspect, membrane (208) is microfabricated, e.g. from a siliconsubstrate, using conventional micromachining techniques described in theabove references. Typically, the diameters of orifices (209) are 25 to35 percent of the expected diameters of particles (211). It is importantthat the aqueous solution (200) does not wet the membrane surfaces,particularly in and/or at the orifices so that the droplets or micellesentering the continuous phase have a quick break off from the rest ofthe aqueous phase.

In another aspect, droplets of gel reaction mixture (less initiator) maybe formed then polymerized by exposure to initiator and heat in a batchmode by at least the following methods. In particular, monodispersepolyacrylamide particles may be made by first producing an emulsion withmonodisperse droplets of polyacrylamide reaction mixture without aninitiator followed by combining with either a second emulsion with aninitiator in the disperse phase or a continuous phase (equivalent tothat of the first emulsion) saturated with an initiator. Method 1. Amonodisperse emulsion is mixed with a micro or macro emulsion ofinitiator in the same oil/surfactant system. The initiator emulsion isgenerated rapidly by vortexing or using an Ultra Turrax, or likeimmiscible phase fluid. Since the monomers are soluble in the continuousphase polymerization of monomer in the initiator emulsion has beenobserved. Method 2. An initiator soluble in the continuous phase is usedto initiate the polymerization in a previously generated emulsion. Useof oil-soluble initiators is well-known in the art as evidenced by thefollowing references, which are incorporated by references: Alduncin etal, Macromolecules, 27: 2256-2261 (1994); Capek, Adv. Colloid andInterface Science, 91: 295-334 (2001); Gromov et al, Vysokomol. Soyed.A30: 1164-1168 (1988); U.S. Pat. No. 3,284,393. The pre-made emulsion issimply diluted with a saturated solution of initiator (for example, a1:1 ratio of emulsion to initiator solution in a polyacrylamide system)and heated to a temperature up to or below 96° C. In one aspect ofmaking polyacrylamide nucleic acid polymer particles, the emulsion isheated to 90° C. for 2 h. Alternatively a water soluble initiator can beused which will not require the dilution of the emulsion, or the oilused during the formation of the monodisperse emulsion can be saturatedwith an appropriate initiator. For method 1, only water solubleinitiators would be used which includes azo type compounds as well asinorganic peroxides. Exemplary initiators include ammonium persulfate,hydroxymethanesulfinic acid monosodium salt dihydrate, potassiumpersulfate, sodium persulfate, and the like. For methods 2. both watersoluble (in dispersed phase) or oil soluble initiators may be used.Example of each are given below in Tables I and II. Note: Thetemperature at which the initiator has a half-life of 10 h is given. Thelower the temperature the more reactive the initiator. Selection ofappropriately reactive initiator allows one of ordinary skill in the artto tune the rate at which the polymerization is initiated and whatmaximum temperatures are used during the reaction.

This approach is illustrated in FIG. 2B. Nucleic acid polymer particlesmay be made in a batch-mode process where a polymerization initiator isintroduced after droplets of gel reaction mixture are formed. A similarmembrane emulsion apparatus (248) as described in FIG. 2A is used togenerate spheroidal droplets of gel reaction mixture (249) in animmiscible continuous phase (202), except that gel reaction mixture inthis embodiment does not contain a polymerization initiator. Afterleaving apparatus (248), continuous phase and droplets (250) are placedin tube (252), after which the mixture is centrifuged (254) to drivedroplets (255) to the bottom of the tube and supernatant (257)comprising the continuous phase is removed (256). Droplets (253) arethen resuspended (258) in continuous phase fluid that contains asuitable initiator, after which the mixture is immediately placed (260)in oven (261) where it is heated to drive the polymerization reaction.After polymerization is complete, the mixture is cooled and washed (262)several times to remove the continuous phase material from the resultingnucleic acid polymer particles.

In one aspect, the invention include a method of making nucleic acidpolymer particles comprising the following steps: (a) combining in areaction mixture hydrophilic monomers each having at least one reactivefunctionality and at least one complementary functionality andhydrophilic cross-linkers each having at least two of either thereactive functionality or complementary functionality, (i) wherein thereactive functionality and complementary functionality are capable ofreacting with one another under catalytic conditions to form covalentlinkages, (ii) wherein a proportion of the monomer have an ancillaryfunctionality or an oligonucleotide attached, the ancillaryfunctionality being capable of reacting with a capture moiety withoutcross reacting with the reactive functionality or the complementaryfunctionality, and (iii) wherein concentrations of the monomers andcross-linkers, and the proportion of monomers having an ancillaryfunctionality or oligonucleotide in the reaction mixture are selected sothat a cross-linked polymer is capable of forming that has a density ofoligonucleotides or ancillary functionalities of at least 1×10⁵ per μm³and an average pore size in the range of from 20 to 150 nm; (b) passingthe reaction mixture through a porous membrane into a non-aqueous phaseso that spherical droplets of reaction mixture are dispersed into thenon-aqueous phase; and (c) subjecting the spherical droplets of thereaction mixture to catalytic conditions so that polymer networks areformed. Ancillary functionalities include monomer derivatives thatinclude reactive groups such as thiol or bromoacetyl groups disclosed byGhosh et al (cited above), groups comprising one of a pair of clickchemistry reactants, or the like In one aspect, the porous membranecomprises a silicon membrane having a plurality of identical orificesthrough which the reaction mixture passes. In another aspect, the methodincludes a further step of removing the polymer networks from saidnon-aqueous phase and washing the polymer networks. In one embodiment,the hydrophilic monomer is an acrylamide and the hydrophiliccross-linker is an N,N′-methylenebisacylamide. In another embodiment, aproportion of the hydrophilic monomer is an acrydite oligonucleotide.

In another aspect, the invention includes a method of makingmonodisperse populations of polyacrylamide particles comprising the stepof combining a monodisperse emulsion of a polyacrylamide reactionmixture without an initiator and an emulsion with a dispersed phasecontaining an initiator or a continuous phase solution saturated with aninitiator. In one embodiment such reaction mixture includes a nucleicacid acrydite monomer.

TABLE I Water Soluble Azo Initiator Compounds 10 hour half-lifeStructure decomposition temperature

44° C.

47° C.

56° C.

57° C.

60° C.

61° C.

67° C.

80° C.

87° C.

TABLE II Oil Soluble Azo Initiator Compounds 10 hour half-lifedecomposition Structure temperature

 30° C.

 51° C.

 66° C.

 67° C.

 88° C.

 96° C.

104° C.

110° C.

111° C.

Measuring Size Distributions of Nucleic Acid Polymer Particles

In one aspect, size distributions of bulk manufactured polymer networksand/or nucleic acid polymer particles are controlled so that theircoefficients of variation are as small as possible. For such control, itis important to be able to conveniently measure the sizes of a sample ofcandidate particles to determine whether their populations haveappropriate coefficients of variation. Many techniques are available formaking such measurements, including laser diffraction, flow cytometry,coulter counting, image analysis, acoustical spectroscopy, and the like.Instruments for laser diffraction are commercially available, e.g.Malvern Instruments (Malvern, United Kingdom); instruments for flowanalysis are commercially available from Becton Dickinson (San Jose,Calif.); Image analysis systems and software are widely availablecommercially, e.g. Becton Dickinson, BioImaging Systems (Rockville,Md.). The foregoing techniques for characterizing particles aredisclosed in Dukhin and Goetz, Ultrasound for Characterizing Colloids(Elsevier Science, 2002); Elaissari, editor, Colloidal Polymers:Synthesis and Characterization (Marcel Dekker, Inc., New York, 2003);Shapiro, Practical Flow Cytometry, 4^(th) edition (Wiley-Liss, 2003);and like references. In the case of polymer networks comprisingpolyacrylamide, fluorescent monomers are available that may be added togel reaction mixtures for incorporation into the polymer networks to aidin their tracking and sizing, e.g. U.S. Pat. No. 5,043,406.

Making Amplicon Libraries with Nucleic Acid Polymer Particles

Nucleic acid polymer particles of the invention are particularly usefulin multiplex genetic assays, including analysis of single nucleotidepolymorphisms, DNA sequencing, and the like, where polynucleotideanalytes, i.e. target polynucleotides, in a sample must be amplified inthe course of analysis. Such analytical techniques use a wide variety ofamplification methodologies which can be used with nucleic acid polymerparticles of the invention, including, but not limited to, emulsion PCR(cmPCR), bridge amplification, NASBA, rolling circle amplification, andthe like. Exemplary references disclosing such techniques are describedin the following references, which are incorporated by reference:Marguiles et al, Nature, 437: 376-380 (2005); Adams et al, U.S. Pat. No.5,641,658; Boles et al, U.S. Pat. No. 6,300,070; Berka et al, U.S.patent publication 2005/0079510; Shapero et al (cited above); and thelike.

Bridge PCR amplification on surfaces is described in Adessi et al (citedabove) and in Boles et al, U.S. Pat. No. 6,300,070, which isincorporated by reference. Briefly, the technique is illustrated in FIG.3. A substrate (300) is provided that has attached via their 5′ ends atleast two primer sequences, A (302) and B (304). Template (306) having3′ primer binding site A′ (308) (that is complementary to A) and primersequence A (310) (that has the same sequence as B on surface (300))anneals to a primer A on surface (300) so that primer A may be extended,e.g. by a polymerase, along template (306) to produce double strandedproduct (316). Template (306) is melted off (314) leaving singlestranded extention product (318) attached to surface (300). Conditionsare applied so that single stranded extension product (318) anneals toan adjacent primer B (302) on surface (300) so that such primer B may beextended (320) to form extension product (322). After melting, extensionproducts (318) and (322) are available (324) for additional cycles ofannealing and extension which form populations of extension productshaving identical sequences to (318) and (322). In some embodiments, oneof primers A and B may have a scissile linkage for its removal to obtaina single population on surface (300).

In accordance with a method of the invention, a bridge PCR may beperformed on nucleic acid polymer particles described herein. The methodmay be employed to make amplicon libraries without the use of emulsionreactions. As illustrated in FIG. 4, suspension (400) of nucleic acidpolymer particles (402) is combined with template (404), the latterbeing in a very dilute concentration relative to the concentration ofnucleic acid polymer particles, so that the probability of two differenttemplates annealing to the same nucleic acid polymer particle is verylow. On a molar basis, for example, nucleic acid polymer particle may bepresent at 10 times, or 100 times the concentration of template. As inFIG. 3, template (404) has primer binding region at its 5′ end (406)that is complementary to one (408) of two primers on the nucleic acidpolymer particles and a sequence at its 3′ end (409) identical to thatof the other primer on the nucleic acid polymer particles. Aftertemplate (404) anneals to primer (408), primer (408) is extended to formextension product (410), after which template (404) is melted andreleased (414). Extension product (410) may then anneal to other primerson the nucleic acid polymer particle to form additional extensionproducts and eventually a bi-clonal population (412) of templates andits complement in reverse orientation. As released template (404) mayparticipate in further amplifications on other nucleic acid polymerparticles, preferably the spacing or concentration of such particles iscontrolled to reduce the probability that such an event occurs (whichotherwise may cross contaminate another particle with a second templateamplicon). In particular, whenever handling or operations result innucleic acid polymer particles settling out of solution, a closelypacked mass of particles forms, which could facilitate such crosscontamination. As illustrated in FIG. 5, this may be prevented orreduced by including inert spacer particles (500) along with nucleicacid polymer particles (502). In one embodiment, the number and size ofspacing particles (500) may be selected to control expected distance(504) between nucleic acid polymer particles (502). In anotherembodiment, for nucleic acid polymer particles and spacer particles ofapproximately the same size, a ratio of spacer particle to nucleic acidpolymer particle is 10:1, or 100:1, or 1000:1. Spacer particles may alsobe selected that are smaller in size than nucleic acid polymer particlesso that interstitial spaces between them have smaller cross sections andcreate longer diffusion paths. In one embodiment, spacer particles maybe swellable so that interstitial spaces are reduced or eliminated uponswelling. Spacer particles may also contain tethered nucleases fordigesting released or unused surplus templates that remain in thereaction solution. Compositions and techniques for making selecting andmaking spacer particles with covalently attached or trapped nucleasesare described in Hermanson, Bioconjugate Techniques, 2^(nd) edition(Academic Press); and like references.

In one aspect, the invention provides a method of making an ampliconlibrary comprising the steps of: (a) combining in a polymerase chainreaction mixture a library of polynucleotide fragments each having afirst primer binding site at one end and a second primer binding site atthe other end, and a population of nucleic acid polymer particles eachcomprising a non-nucleosidic polymer network having attached thereto afirst primer and a second primer each at a concentration of at least1×10⁵primers per μm³ such that each polynucleotide fragment is capableof annealing to a first primer by its first primer binding site and to asecond primer by a complement of its second primer binding site; and (b)performing a polymerase chain reaction in the presence of a quantity ofspacer particles so that primers of the polymer networks are extendedalong polynucleotide fragments annealed thereto so that clonalpopulations of complements of such polynucleotide fragments are formedon the polymer networks, thereby forming an amplicon library, thequantity of spacer particles being selected to preventcross-contamination of amplicons. In one aspect, the polymer networkseach have a volume and a concentration of the polynucleotide fragmentsand a concentration of said polymer networks are selected so that insaid step of combining at least 10 percent of said polymer networks haveat a single said polynucleotide fragment within its volume. For someembodiments, the non-nucleosidic polymer network has a volume of lessthan 1.4×10⁴ μm³.

In still another aspect, the invention provides a method of making anamplicon library by performing a bridge polymerase chain reaction on acomposition of monodisperse nucleic acid polymer particles. In anembodiment of such method the composition of monodisperse nucleic acidpolymer particles includes a quantity of spacer particles.

Alternatively, nucleic acid polymer particles being used in bridge PCRmay be amplified in a thermocycler instrument that provides agitation orrotation of the reaction chambers or tubes to present settling orprolonged particle-particle contact. A simple device shown in FIG. 7 maybe used as such a thermocycler. Reaction tubes (not shown) are placed inholders near the outer periphery of wheel (702) which may be loweredinto heated oil bath (700) and rotated by motor (704). Wheel (702)rotates at a predetermined speed and depth in oil bath (700), whosetemperature is controlled by controller (710) by way of thermometer(708) and heater (706). By programming controller (710) a thermocycleris provided, which ensures that nucleic acid polymer particles do notsettle during amplification.

Methods of making amplicon libraries may also include a step ofenriching nucleic acid polymer particles having clonal populations ofpolynucleotide fragments. In one embodiment, such enrichment may beaccomplished by affinity purification, for example, by annealing anoligonucleotide with a capture moiety, such as biotin, to a primerbinding site of the polynucleotide fragments, after which the resultingcomplexes may be captured, e.g. by streptavidinated magnetic beads, andseparated from particles without polynucleotide fragments. In anotherembodiment, nucleic acid polymer particles having clonal populations ofpolynucleotide fragments may be separated from particles withoutpolynucleotide fragments by electrophoresis, e.g. using a commerciallyavailable instrument (such as, PippinPrep automated prep gel system,Sage Science, Beverly, Mass.).

Nucleic acid polymer particles may further be used in analysis ofselected sets of genes or other polynucleotide sequences. Sets of suchparticles with specificities for particular predetermined polynucleotidetargets are readily prepared from a single batch of particles using thetechnique outlined in FIG. 6. To a batch of nucleic acid polymerparticles (600) that have the same primers (608) attached by their 5′ends, is annealed adaptor oligonucleotide (602) that comprises 3′ end(604) that is complementary to primer (608) and that includes 5′ end(606) which has a sequence identical to a target polynucleotide to becaptured and amplified. After such annealing, polymerase (610) is addedin a conventional polymerase reaction mixture so that primer (608) isextended along 5′ end of oligonucleotide (602) as a template. After suchextension (614), oligonucleotide (602) is released to leavesequence-specific primer (616) on nucleic acid polymer particle (600).In the same reaction (by providing oligonucleotide (602) as a mixture ofA and B primers) or in a subsequent reaction, the same steps may befollowed to add a second primer for bridge amplification on theresulting set of nucleic acid polymer particles. Alternatively, primer(608) (or a second primer) may be extended in a template-driven ligationreaction, where a 5′ phosphorylated oligonucleotide (not shown)complementary to 5′ segment (606) is provided. This technique may beused to prepare a set of nucleic acid polymer particles containing aplurality of particles each with a different specificity. Such a set maybe used to selectively amplify a predetermined set of targetpolynucleotides in a bridge PCR for analysis.

Nucleic Acid Sequencing with Nucleic Acid Polymer Particles

In one aspect, the invention may be used for carrying out label-free DNAsequencing, and in particular, pH-based DNA sequencing. The concept oflabel-free DNA sequencing, including pH-based DNA sequencing, has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006);and the like. Briefly, in pH-based DNA sequencing, base incorporationsare determined by measuring hydrogen ions that are generated as naturalbyproducts of polymerase-catalyzed extension reactions. Nucleic acidpolymer particles are used advantageously in pH-based sequencing becausegreater concentrations of templates may be attached to them therebyincreasing the signal-to-noise ratio of the pH signal associated withbase incorporations. Nucleic acid polymer particles are used to makeamplicon libraries as described above which, in turn, are used withapparatus as described in Rothberg et at (cited above). In oneembodiment, templates each having a primer and polymerase operably boundare loaded into reaction chambers (such as the microwells disclosed inRothberg et al, cited above), after which repeated cycles ofdeoxynucleoside triphosphate (dNTP) addition and washing are carriedout. In some embodiments, such templates may be attached as clonalpopulations to a solid support, such as a microparticle, bead, or thelike, and such clonal popultations are loaded into reaction chambers.For example, templates may be prepared as disclosed in U.S. Pat. No.7,323,305, which is incorporated by reference. As used herein, “operablybound” means that a primer is annealed to a template so that theprimer's 3′ end may be extended by a polymerase and that a polymerase isbound to such primer-template duplex, or in close proximity thereof sothat binding and/or extension takes place whenever dNTPs are added. Ineach addition step of the cycle, the polymerase extends the primer byincorporating added dNTP only if the next base in the template is thecomplement of the added dNTP. If there is one complementary base, thereis one incorporation, if two, there are two incorporations, if three,there are three incorporations, and so on. With each such incorporationthere is a hydrogen ion released, and collectively a population oftemplates releasing hydrogen ions changes the local pH of the reactionchamber. The production of hydrogen ions is monotonically related to thenumber of contiguous complementary bases in the template (as well as thetotal number of template molecules with primer and polymerase thatparticipate in an extension reaction). Thus, when there is a number ofcontiguous identical complementary bases in the template (i.e. ahomopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, is proportional to thenumber of contiguous identical complementary bases. (The correspondingoutput signals are sometimes referred to as “1-mer”, “2-mer”, “3-mer”output signals, and so on). If the next base in the template is notcomplementary to the added dNTP, then no incorporation occurs and nohydrogen ion is released (in which case, the output signal is sometimesreferred to as a “0-mer” output signal.) In each wash step of the cycle,an unbuffered wash solution at a predetermined pH is used to remove thedNTP of the previous step in order to prevent misincorporations in latercycles. Usually, the four different kinds of dNTP are added sequentiallyto the reaction chambers, so that each reaction is exposed to the fourdifferent dNTPs one at a time, such as in the following sequence: dATP,dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposurefollowed by a wash step. The process is illustrated in FIG. 8 fortemplate (882) with primer binding site (881) attached to nucleic acidpolymer particle (880). Primer (884) and DNA polymerase (886) operablybound to template (882). Upon the addition (888) of dNTP (shown asdATP), polymerase (886) incorporates a nucleotide since “T” is the nextnucleotide in template (882). Wash step (890) follows, after which thenext dNTP (dCTP) is added (892). Optionally, after each step of adding adNTP, an additional step may be performed wherein the reaction chambersare treated with a dNTP-destroying agent, such as apyrase, to eliminateany residual dNTPs remaining in the chamber, which may result inspurious extensions in subsequent cycles.

In one embodiment, a sequencing method exemplified in FIG. 8 may becarry out using the apparatus of the invention in the following steps:(a) disposing a plurality of template nucleic acids into a plurality ofreaction chambers disposed on a sensor array, the sensor arraycomprising a plurality of sensors and each reaction chamber beingdisposed on and in a sensing relationship with at least one sensorconfigured to provide at least one output signal representing asequencing reaction byproduct proximate thereto, and wherein each of thetemplate nucleic acids is hybridized to a sequencing primer and is boundto a polymerase; (b) introducing a known nucleotide triphosphate intothe reaction chambers; (c) detecting incorporation at a 3′ end of thesequencing primer of one or more nucleotide triphosphates by asequencing reaction byproduct if such one or more nucleotidetriphosphates are complementary to corresponding nucleotides in thetemplate nucleic acid; (d) washing unincorporated nucleotidetriphosphates from the reaction chambers; and (e) repeating steps (b)through (d) until the plurality of template nucleic acids are sequenced.For embodiments where hydrogen ion is measured as a reaction byproduct,the reactions further should be conducted under weak buffer conditions,so that the maximum number of hydrogen ions reacts with a sensor and notextraneous components (e.g. microwell or solid supports that may havesurface buffering capacity) or chemical constituents (in particular pHbuffering compounds). In one embodiment, a weak buffer allows detectionof a pH change of at least ±0.1 in said reaction chamber, or at least±0.01 in said reaction chambers.

Example 1 Making Polyacrylamide Nucleic Acid Polymer Particles byMembrane Emulsification

This example describes the method and apparatus for production ofuniformly sized droplets of aqueous solution in non-miscible continuousphase by extrusion through a micro fabricated plate with multiplethrough holes (nozzles, orifices) and the subsequent transformation ofthe emulsion into polymer particles by radical polymerization. Thefabrication of the plate with multiple through holes is described in theabove references.

A solution of specific amounts of acrylamide andmethylene-N,N-bisacrylamide containing a specified concentration ofacrodyte 5′-labled oligonucleotides (primers for PCR) is degassed bybubbling an inert atmosphere (Argon, Nitrogen, Helium) through thesolution for a minimum of 30 minutes. Just prior to emulsification, aradical initiator is added. The radical initiator can be a combinationof ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine(TMED) which catalyses the radical initiation by APS. The amount of TMEDused needs to be carefully adjusted to allow sufficient time foremulsification. APS and V-50 (see below) initiate thermally above ˜65degrees celsius. Alternatively a photoinitiator such2,2′-Azobis(2-methylpropionamidine)dihydrochloride (V-50) may be used inconjunction with a UV light source with strong emission peaks at 220 nmand 365 nm. The aqueous solution above may be dispersed into acontinuous phase via several techniques. For example: The aqueoussolution may be subdivided into droplets by a vibrating membrane withseveral appropriately sized holes (typically 50-70% smaller than theintended diameter of the droplet) after which the droplets are allowedto enter an immiscible continuous phase which may or may not containsurfactants. The droplets may be irradiated with UV prior to enteringthe continuous phase (after leaving the nozzle, in mid air) or afterentering the continuous phase. In one implementation the drops areallowed to polymerize prior to entering the continuous phase which maybe miscible with the un-polymerized dispersed phase. In either case, thehumidity of the atmosphere needs to be controlled to preventuncontrolled evaporative shrinking of the droplets and the atmosphereneeds to be largely oxygen free to allow radical polymerization in thedroplets. An alternative emulsification technique can be described asfollows: The aqueous phase is pumped through a porous membrane (withuniformly sized pores) into an immiscible continuous phase. Afterpolymerization, the beads are recovered from the continuous phase byeither breaking the emulsion (by addition of n-butanol, n-propanoli-propanol or other appropriate chemicals) followed by centrifugation topellet the beads in the bottom of the eppendorf tube or filtrationthrough an appropriately sized filter. After washing with an appropriatebuffer, the beads can be used for PCR amplification of DNA libraryelements or direct hybridization of DNA fragments with the reversecomplement to the attached oligonucleotide.

Example 2 Making Polyacrylamide Nucleic Acid Polymer Particles byMembrane Emulsification with Batch Mode Initiation

This example describes membrane emulsification and the subsequenttransformation of the aqueous micelles of the emulsion intopolyacrylamide particles by radical polymerization in batch mode usingan initiator-saturated oil phase. The steps of the process comprise (a)formation of a gel reaction mixture-in-oil emulsion using a membrane,(b) particle polymerization, and (c) particle extraction and washing.

The following reagents are employed in the process: (a) SNAPP Oilcomprises the following mixture: Tegosoft™ DEC oil (730 mL), ABIL WE09(70 gm), and mineral oil (200 mL) (SNAPP oil is stored under argon); (b)SNAPP Buffer: 1× TE, 0.1% Triton X-100, 0.02% sodium azide; (c)Acrylamide Solution: 50 mg N,N-methylene bisacrylamidc, 450 mgacrylamide, 550 uL double distilled H₂O kept under argon; (d) DNA Mix:10 umol 30-mer acrydite oligonucleotide with 18 C spacer in 2.5 mL GelReaction Mixture is formed by mixing the following together under argonfor a total volume of 1400 uL: 526.4 uL DNA Mix, 14 mL TMED, 299.6 mLH₂O, and 560 mL Acrylamide Solution. Under argon, 1 mL of the GelReaction Mixture is added to the upper compartment of a two-compartmentrig (see FIGS. 2A & 2B) having an emulsification membrane dividing theupper compartment from the lower compartment, so that the Gel ReactionMixture flows under gravity from the upper compartment through theemulsification membrane (thereby forming droplets or micelles) into aflow of SNAPP Oil in the lower compartment. SNAPP Oil is driven into thelower chamber at a rate of 2.4 mL/hr. About 50 mL of the SNAPP Oil-GelReaction Mixture emulsion is collected in a centrifuge tube, after whichit is centrifuged so that the Gel Reaction Mixture micelles are drivento the bottom. All but about 1 mL of the supernatant SNAPP Oil isremoved, after which the micelles are resuspended by adding 20 mL ofinitiator (1,1′-azobis(cyclohexanecarbonitrile))-saturated SNAPP Oil.(Initiator-saturated SNAPP Oil is made by mixing 500 mg initiator in 25mL SNAPP Oil under argon with vigorous mixing). The resuspended micellesundergo polymerization by placing them in an oven at 90° C. under argonand constant rotation for 2 hr. and 2 min, after which they are removedand immediately place in a 4° C. refrigerator for at least 1 hr. TheSNAPP Oil is removed from the polymerized particles by centrifuging toform a pellet followed by resuspension in butanol with vortexing, andthen repeating, after which the polymerized particles are resuspended in0.1% SDS and sonicated for 3 min. The polymerized particles are thentwice centrifuged, resuspended in SNAPP Buffer, and sonicated for 3 min,after which they are resuspended in SNAPP Buffer and stored at 4° C.

The size distribution of the above nucleic acid polymer particles may bemeasured using a Guava flow cytometer after hybridizing a labeledoligonucleotide with a sequence complementary to at least one of thoseof the nucleic acid polymer particle. An exemplary protocol is asfollows: (1) suspend about 5 million particles in 9 μL 1× PBS 0.2%Tween, (2) add 2 μL of 100 μM biotinylated oligonucleotide complement,(3) anneal at 95° C. for 2 min followed by 37° C. for 2 min, (4)centrifuge to remove supernatant, (5) wash 2× with 1× PBS 0.2% Tween,(6) resuspend in 10 μL 1× PBS 0.2% Tween, (7) add 0.5 μLstreptavidin-FITC (commercial reagent, e.g. Anaspec, Fremont, Calif.),(8) wash 2× with 1× PBS 0.05 Tween, (9) add to 1 mL 1× PBS 0.05% Tween,and (10) run sample on flow cytometer (e.g. EasyCyte mini, GuavaTechnologies).

Size distributions of nucleic acid polymer particles may also bemeasured by staining and counting them using a fluorescent microscopewith automatic particle counting software. A series of dilutions ofnucleic acid polymer particles are stained with a series ofconcentrations of a nucleic acid stain, such as SYBR Gold (Invitrogen),after which they are place in separate wells of multi-welledpoly-1-lysine coated slides (e.g. Tekdon Inc.). A comparison of particlesizing and counting data from flow system and microscope measurementsshows good correlation. CVs of size distributions of samples from abatch of nucleic acid polymer particles, designated B4, was determinedby slide counting (described above) and by flow cytometry counting(described above). For slide counting, three samples of particles (4.32million/uL) were analyzed and coefficients of variation were determinedto be 12.9%, 13.8%, and 9.6%, respectively. A single sample of particles(3.8 million/uL) was analyzed and a coefficient of variation wasdetermined to be 5.88%.

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. The present invention is applicableto a variety of implementations and other subject matter, in addition tothose discussed above.

Definitions

Unless otherwise specifically defined herein, terms and symbols ofnucleic acid chemistry, biochemistry, genetics, and molecular biologyused herein follow those of standard treatises and texts in the field,e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman,N.Y., 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers,New York, 1975); Strachan and Read, Human Molecular Genetics, SecondEdition (Wiley-Liss, New York, 1999).

“Amplicon” means the product of a polynucleotide amplification reaction;that is, a clonal population of polynucleotides, which may be singlestranded or double stranded, which are replicated from one or morestarting sequences. The one or more starting sequences may be one ormore copies of the same sequence, or they may be a mixture of differentsequences that contain a common region that is amplified, for example, aspecific exon sequence present in a mixture of DNA fragments extractedfrom a sample. Preferably, amplicons are formed by the amplification ofa single starting sequence. Amplicons may be produced by a variety ofamplification reactions whose products comprise replicates of the one ormore starting, or target, nucleic acids. In one aspect, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al,U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491(“NASBA”); Lizardi, U.S. Pat. No. 5,854,033; Aono et al, Japanese patentpubl. JP 4-262799 (rolling circle amplification); and the like. In oneaspect, amplicons of the invention are produced by PCRs. As used herein,the term “amplifying” means performing an amplification reaction. An“reaction mixture,” including an “amplification reaction mixture,” meansa solution containing all the necessary reactants for performing areaction, which may include, but not be limited to, buffering agents tomaintain pH at a selected level during a reaction, salts, co-factors,scavengers, and the like. A “solid phase amplicon” means a solid phasesupport, such as a particle or bead, having attached a clonal populationof nucleic acid sequences, which may have been produced by a processsuch as emulsion PCR, or like technique. One aspect of the invention issolid phase amplicons comprising nucleic acid polymer particles. In someembodiments, amplicons may be produced by isothermal reactions, such asrolling circle amplification reactions, NASBAs, or helicase-mediatedamplification reactions, e.g. U.S. Pat. No. 7,282,328, which isincorporated by reference.

“Microwell” which is used interchangeably with “reaction chamber,” meansa special case of a “reaction confinement region,” that is, a physicalor chemical attribute of a solid substrate that permit the localizationof a reaction of interest. Reaction confinement regions may be adiscrete region of a surface of a substrate that specifically binds ananalyte of interest, such as a discrete region with oligonucleotides orantibodies covalently linked to such surface. Usually reactionconfinement regions are hollows or wells having well-defined shapes andvolumes which are manufactured into a substrate. These latter types ofreaction confinement regions are referred to herein as microwells orreaction chambers, and may be fabricated using conventionalmicrofabrication techniques, e.g. as disclosed in the followingreferences: Doering and Nishi, Editors, Handbook of SemiconductorManufacturing Technology, Second Edition (CRC Press, 2007); Saliterman,Fundamentals of BioMEMS and Medical Microdevices (SPIE Publications,2006); Elwenspoek et al, Silicon Micromachining (Cambridge UniversityPress, 2004); and the like. Preferable configurations (e.g. spacing,shape and volumes) of microwells or reaction chambers are disclosed inRothberg et al, U.S. patent publication 2009/0127589; Rothberg et al,U.K. patent application GB24611127, which are incorporated by reference.Microwells may have square, rectangular, or octagonal cross sections andbe arranged as a rectilinear array on a surface. Microwells may alsohave hexagonal cross sections and be arranged as a hexagonal array,which permit a higher density of microwells per unit area in comparisonto rectilinear arrays. Exemplary configurations of microwells are asfollows: In some embodiments, the reaction chamber array comprises 10²,10³, 10⁴, 10⁵, 10⁶ or 10⁷ reaction chambers. As used herein, an array isa planar arrangement of elements such as sensors or wells. The array maybe one or two dimensional. A one dimensional array is an array havingone column (or row) of elements in the first dimension and a pluralityof columns (or rows) in the second dimension. The number of columns (orrows) in the first and second dimensions may or may not be the same.Preferably, the array comprises at least 100,000 chambers. Preferably,each reaction chamber has a horizontal width and a vertical depth thathas an aspect ratio of about 1:1 or less. Preferably, the pitch betweenthe reaction chambers is no more than about 10 microns. Briefly, in oneembodiment microwell arrays may be fabricated as follows: After thesemiconductor structures of a sensor array are formed, the microwellstructure is applied to such structure on the semiconductor die. Thatis, the microwell structure can be formed right on the die or it may beformed separately and then mounted onto the die, either approach beingacceptable. To form the microwell structure on the die, variousprocesses may be used. For example, the entire die may be spin-coatedwith, for example, a negative photoresist such as Microchem's SU-8 2015or a positive resist/polyimide such as HD Microsystems HD8820, to thedesired height of the microwells. The desired height of the wells (e.g.,about 3-12 μm in the example of one pixel per well, though not solimited as a general matter) in the photoresist layer(s) can be achievedby spinning the appropriate resist at predetermined rates (which can befound by reference to the literature and manufacturer specifications, orempirically), in one or more layers. (Well height typically may beselected in correspondence with the lateral dimension of the sensorpixel, preferably for a nominal 1:1-1.5:1 aspect ratio, height:width ordiameter.) Alternatively, multiple layers of different photoresists maybe applied or another form of dielectric material may be deposited.Various types of chemical vapor deposition may also be used to build upa layer of materials suitable for microwell formation therein. In oneembodiment, microwells are formed in a layer oftetra-methyl-ortho-silicate (TEOS). The invention encompasses anapparatus comprising at least one two-dimensional array of reactionchambers, wherein each reaction chamber is coupled to achemically-sensitive field effect transistor (“chemFET”) and eachreaction chamber is no greater than 10 μm³ (i.e., 1 pL) in volume.Preferably, each reaction chamber is no greater than 0.34 pL, and morepreferably no greater than 0.096 pL or even 0.012 pL in volume. Areaction chamber can optionally be 2², 3², 4², 5², 6², 7², 8², 9², or10² square microns in cross-sectional area at the top. Preferably, thearray has at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or morereaction chambers. The reaction chambers may be capacitively coupled tothe chemFETs, and preferably are capacitively coupled to the chemFETs.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C. The term “PCR” encompasses derivativeforms of the reaction, including but not limited to, RT-PCR, real-timePCR, nested PCR, quantitative PCR, multiplexed PCR, concatemeric PCR,and the like. Reaction volumes range from a few hundred nanoliters, e.g.200 nL, to a few hundred ρL, e.g. 200 μL.

“Polymer network” means a structure comprising covalently connectedsubunits (monomers, crosslinkers, and the like) in which all suchsubunits are connected to every other subunit by many paths through thepolymer phase, and wherein there are enough polymer chains bondedtogether (either physically or chemically) such that at least one largemolecule is coextensive with the polymer phase (i.e. the structure isabove its gel point). Preferably a polymer network has a volume in therange of from 65 aL to 15 pL, or from 1 fL to 1 pL.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their internucleosidic linkagesmay be naturally occurring or may be analogs thereof, e.g. naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moities, or bases at any or some positions.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al, Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2^(nd) Edition (Cold SpringHarbor Press, New York, 2003).

“Sample” in one aspect means a quantity of material from a biological,environmental, medical, or patient source in which detection ormeasurement of one or more analytes is sought. A sample may also includea specimen of synthetic origin. Biological samples may be animal,including human, fluid, solid (e.g., stool) or tissue, as well as liquidand solid food and feed products and ingredients such as dairy items,vegetables, meat and meat by-products, and waste. Biological samples mayinclude materials taken from a patient including, but not limited tocultures, blood, saliva, cerebral spinal fluid, needle aspirates, andthe like. Biological samples also may be obtained from animals.Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, and the like. In another aspect, “sample” means a material orsubstance extracted, partially purified, separated, or otherwiseobtained by sample preparation techniques from a sample as defined inthe previous sentences (collectively referred to as “extractedmaterial”). Such extracted materials that are occasionally referred toherein as “samples” include but are not limited to nucleic acids (forexample, DNA or RNA extracted material), protein extracted material,lipid extracted material, and the like.

1. An amplicon library comprising a plurality of amplicons, each amplicon comprising a clonal population of a single polynucleotide from a nucleic acid library, each polynucleotide of the clonal population being attached to a non-nucleosidic polymer network, each such polymer network having a volume and the polynucleotides of the clonal population being attached to the polymer network throughout its volume, wherein the number of attached polynucleotides is at least 6.9×10⁴ per μm³.
 2. The amplicon library of claim 1 wherein said volumes of said non-nucleosidic polymer network are each less than 1.4×10⁴ μm³ and have a coefficient of variation of fifteen percent or less.
 3. The amplicon library of claim 2 wherein said volumes of said non-nucleosidic polymer network are spheroidal and are each less than 1.8×10³ μm³.
 3. The amplicon library of claim 1 wherein said volumes of said polymer networks are spheroidal.
 4. The amplicon library of claim 1 wherein said polymer networks are each polyacrylamide gels having a gram/milliliter percent polyacrylamide composition of 15 percent or less.
 5. The amplicon library of claim 1 wherein said polynucleotides each have a size in the range of 100-1200 nucleotides.
 6. The amplicon library of claim 5 wherein said polynucleotides of said clonal populations are attached to said polymer networks randomly throughout their respective volumes.
 7. The amplicon library of claim 1 wherein said polymer networks are polyacrylamide gels having a gram/milliliter percent polyacrylamide composition and a acrylamide/bis-acrylamide percent composition that provides a porosity permitting substantially free diffusion of a DNA polymerase.
 8. A method of making an amplicon library comprising the steps of: combining in an amplification reaction mixture a library of polynucleotide fragments each having at least one primer binding site and a population of non-nucleosidic polymer networks, each such polymer network having a volume of less than 1.4×10⁴ μm³ and having primers attached thereto, and the volumes of the non-nucleosidic polymer networks having a coefficient of variation of fifteen percent or less; forming an emulsion having a dispersed aqueous phase of micelles such that at least one micelle contains at most one polymer network and one polynucleotide fragment; and performing an amplification reaction in the micelles so that primers of the polymer networks are each extended along a polynucleotide fragment annealed thereto so that clonal populations of complements of such polynucleotide fragments are formed on the polymer networks, thereby forming an amplicon library.
 9. The method of claim 8 wherein said amplification reaction is a polymerase chain reaction.
 10. The method of claim 8 wherein said amplification reaction is an isothermal amplification reaction.
 11. The method of claim 10 wherein said polynucleotide fragments have a size in the range of from 150 to 500 nucleotides.
 12. A method of making an amplicon library comprising the steps of: combining in a polymerase chain reaction mixture a library of polynucleotide fragments each having a first primer binding site at one end and a second primer binding site at the other end, and a population of nucleic acid polymer particles each comprising a non-nucleosidic polymer network having attached thereto a first primer and a second primer such that each polynucleotide fragment is capable of annealing to a first primer by its first primer binding site and to a second primer by a complement of its second primer binding site; performing a polymerase chain reaction so that primers of the polymer networks are extended along polynucleotide fragments annealed thereto so that clonal populations of complements of such polynucleotide fragments are formed on the polymer networks, thereby forming an amplicon library.
 13. The method of claim 12 wherein each of said polymer networks has a volume and wherein a concentration of said polynucleotide fragments and a concentration of said polymer networks are selected so that in said step of combining at least 10 percent of said polymer networks have at a single said polynucleotide fragment within its volume.
 14. The method of making an amplicon library of claim 12 wherein said non-nucleosidic polymer network has a volume of less than 1.4×10⁴ μm³ and the volumes of said non-nucleosidic polymer networks have a coefficient of variation of fifteen percent or less.
 15. A method of determining a sequence of a nucleic acid, the method comprising the steps of: disposing an amplicon library in a planar array, the amplicon library comprising a plurality of amplicons, each amplicon comprising a clonal population of a single polynucleotide from a nucleic acid library, each polynucleotide of the clonal population being attached to a non-nucleosidic polymer network, each such polymer network having a volume and the polynucleotides of the clonal population being attached to the polymer network throughout its volume, wherein the number of attached polynucleotides is at least 6.9×10⁴ per μm³ and the volumes of the polymer networks have a coefficient of variation of fifteen percent or less. performing a sequencing reaction simultaneously on the fragments of the amplicons of the library.
 16. The method of claim 15 wherein said planar array is an array of sample retaining regions such that substantially each sample retaining region has at most one amplicon.
 17. The method of claim 16 wherein said array of sample retaining regions is a microwell array of regularly spaced microwells having a pitch therebetween of 10 μm or less.
 18. The method of claim 17 wherein said microwell array is disposed on an integrated array of chemical field effect transistors in a circuit-supporting substrate, wherein the integrated array has a pitch of 10 μm or less and each microwell is positioned on at least one chemical field effect transistor that is configured to generate at least one output signal related to an amount or concentration of a reaction byproduct generated in a sequencing reaction on an amplicon in such microwell.
 19. A composition comprising: a field effect transistor array in a circuit-supporting substrate, such transistor array having disposed on its surface an array of sample retaining regions such that each sample retaining region is positioned on at least one field effect transistor; and a population of nucleic acid polymer particles randomly distributed in the sample retaining regions, the population comprising a plurality of different nucleic acids and the nucleic acid polymer particles each having a volume and the volumes having a coefficient of variation of fifteen percent or less.
 20. The composition of claim 19 wherein there is at most one said nucleic acid polymer particle per said sample retaining region.
 21. A composition of nucleic acid polymer particles each comprising oligonucleotides attached to a non-nucleosidic polymer network, each such polymer network having a volume and the oligonucleotides being attached to the polymer network throughout its volume, wherein the oligonucleotides have an average nearest neighbor distance in the range of from 10 to 20 nm and the volumes of the non-nucleosidic polymer networks have a coefficient of variation of fifteen percent or less.
 22. The composition of claim 21 wherein said oligonucleotides are primers each attached to said polymer network by its 5′ end.
 23. A method of amplifying a target polynucleotide in a sample, the method comprising the steps of: combining a sample and nucleic acid polymer particles in a polymerase chain reaction mixture, each such particle comprising a non-nucleosidic polymer network having attached throughout its volume a first primer and a second primer each at a concentration of at least 1×10⁵ primers per mm³, such that the target polynucleotide is capable of annealing to the first primer by a first region and to a second primer by a complement of a second region; and performing a polymerase chain reaction so that primers on a nucleic acid polymer particle are repeatedly extended along a target polynucleotide annealed thereto so that a clonal population of complements of such target polynucleotide is formed on the polymer networks. 